Elevated temperature and carbon dioxide levels alter growth rates and shell composition in the fluted giant clam, Tridacna squamosa

Giant clams produce massive calcified shells with important biological (e.g., defensive) and ecological (e.g., habitat-forming) properties. Whereas elevated seawater temperature is known to alter giant clam shell structure, no study has examined the effects of a simultaneous increase in seawater temperature and partial pressure of carbon dioxide (pCO2) on shell mineralogical composition in these species. We investigated the effects of 60-days exposure to end-of-the-century projections for seawater temperature (+ 3 °C) and pCO2 (+ 500 µatm) on growth, mineralogy, and organic content of shells and scutes in juvenile Tridacna squamosa giant clams. Elevated temperature had no effect on growth rates or organic content, but did increase shell [24Mg]/[40Ca] as well as [40Ca] in newly-formed scutes. Elevated pCO2 increased shell growth and whole animal mass gain. In addition, we report the first evidence of an effect of elevated pCO2 on element/Ca ratios in giant clam shells, with significantly increased [137Ba]/[40Ca] in newly-formed shells. Simultaneous exposure to both drivers greatly increased inter-individual variation in mineral concentrations and resulted in reduced shell N-content which may signal the onset of physiological stress. Overall, our results indicate a greater influence of pCO2 on shell mineralogy in giant clams than previously recognized.

Giant clams (Tridacninae) inhabit nutrient-poor tropical coral reefs where they live in symbiosis with photosynthetic dinoflagellates of the family Symbiodinaceae. Within these biomes, giant clams are ecologically important species 1 whose large shells provide habitat for numerous encrusting epibiota and which play a significant role in carbonate deposition/liberation 2 and reef formation 1 . Because the strength and durability of clam shells depends on their elemental composition, significant research effort has been devoted to shedding light on the effects of environmental drivers on shell isotopic composition in giant clams [3][4][5][6][7][8] . In addition, as shell composition has the potential to provide useful information about conditions during biomineralization, there has been great interest in the influence of abiotic and biotic effects on incorporation of elemental impurities, especially Group II elements such as 24 Mg, 88 Sr, and 137 Ba, which substitute for 40 Ca within the calcium carbonate (CaCO 3 ) lattice of giant clams' shells 4,[8][9][10][11] .
In contrast to scleractinian corals, for which environmental influences on carbonate element/Ca ratios are well-characterized(e.g., 12 ), our current understanding of the abiotic controls on trace element incorporation in Tridacna clams remains equivocal (see Table S1). Although shell [ 24 Ca] ratios of giant clams appear strongly influenced by seawater temperature, and are even used as proxies for past sea surface temperature (SST) 4 , the exact nature of this influence, including its directionality, remains unclear 11 . In addition to this ambiguity regarding the influence of temperature, relatively few studies have examined the effects of multiple environmental drivers acting simultaneously on shell mineral structure in giant clams 13 , and none have investigated the effect of anthropogenically-driven increases in the partial pressure of carbon dioxide (pCO 2 ) in seawater (i.e., ocean acidification) on element/Ca ratios in tridacnines. As alterations in shell mineral composition can have significant ecological impacts during a clam's life (e.g., through reduced shell strength and thus increased vulnerability to crushing predators), as well as important repercussions for the durability and persistence of Tridacna-derived carbonate reefs, investigating mineralogical changes in response to multifaceted environmental change is an important first step for improving our understanding of current and projected global change impacts on giant clams and their associated reef habitats.
Knowledge of giant clam physiological responses to multiple climate-change stressors, including ocean warming and acidification, is vital for informing conservation strategies under rapid and ongoing environmental change 14 . Previous research has shown that exposure to elevated seawater temperature has profound impacts on giant clam physiology including decreased fertilization success 15 , reduced photosymbiont density 16,17 , altered lipid biosynthesis 18 , accumulation of reactive oxygen species 18 , altered holobiont oxygenic/respiratory balance 19,20 , and increased juvenile mortality 21 . Similarly, at elevated seawater pCO 2 , giant clams have been shown to grow more slowly 22 , build smaller shells 13,22,23 , and suffer higher mortality 21 than under present-day ocean conditions 21,22 . Simultaneous prolonged (41 days) exposure to elevated pCO 2 and elevated temperature reduced calcification rates and altered shell ultrastructure, increasing disordered crystalline lamellae 13 . Whereas these changes in shell crystalline structure suggest underlying alterations in shell mineral and organic content, no previous study has explicitly investigated paired mineralogical and organic responses in giant clam shells. In this study, we investigated the effects of elevated seawater temperature and pCO 2 on the mineral composition and organic content of shells of the fluted giant clam Tridacna squamosa (Lamarck, 1819) to improve our understanding of physiological mechanisms related to reduced calcification in giant clams under projected future ocean conditions. Because elevated seawater pCO 2 results in decreased bioavailability of carbonate ions, we hypothesized that extended exposure of juvenile T. squamosa to elevated pCO 2 would result in decreased shell growth rates and increased incorporation of trace mineral elemental impurities. Similarly, because elevated seawater temperature can result in holobiont stress (e.g., symbiotic breakdown and bleaching 24 ) and alter trace element incorporation during biomineralization (Table S1), we hypothesized that exposure to elevated temperature would also decrease shell growth rates and alter mineralogical profiles in T. squamosa shells. Finally, we hypothesized that these two environmental drivers would interact synergistically when combined, leading to a greater reduction in shell growth and larger alterations of mineral profiles than when acting in isolation. To test these hypotheses, juvenile fluted giant clams were exposed to current and projected end-twenty-first century seawater conditions for 60 days, following which newly-formed (i.e., formed under treatment) and older-growth (i.e., formed prior to treatment) shell and scute were collected for analysis.

Methods
Specimen collection and exposure conditions. Juveniles of the giant clam Tridacna squamosa selected for this study (N = 32; shell length 35.81 ± 8.20 mm, mean ± s.d.) were spawned at the Darwin Aquaculture Centre (Wickham, Northern Territory, Australia) from wild-caught broodstock collected from the Northern Territory, Australia. Juvenile clams were transferred to the James Cook University aquarium facility where they were kept in natural seawater sourced from the Australian Institute of Marine Science seawater intake facility at Cape Cleveland until the experiment started. This natural seawater was filtered to 1 µm and UV-sterilized before introduction into the aquarium systems. Two > 8000 L recirculating seawater systems were maintained at two different partial pressures of carbon dioxide (pCO 2 ) cross factored with two levels of temperature: + 0.0 and + 3.0 °C. Target values for seawater treatments were selected to mimic present-day and future, end-of-thecentury, global ocean scenarios using the IPCC RCP 8.5 (business-as-usual) projections 25 for temperature (28.5 and 30.5 °C, respectively 26,27 ) and pCO 2 (450 and 950 μatm, respectively 28,29 ). Elevated CO 2 treatments were achieved by dosing 100% CO 2 into a 3000 L temperature-controlled sump on each system to a set pH using a pH control system (AT-Control, Aqua Medic, Germany) following standard techniques (Gattuso et al. 2010). Temperature (C22, Comark, Norwich, U.K.) and pH NBS (HQ40d, Hach, Colorado, U.S.) were recorded daily in the treatment tanks. Realized temperature and CO 2 levels for the four treatment conditions (2 × 2 design) were: (1) control temperature 28.7 °C and control pCO 2 436 µatm; (2) control temperature 28 www.nature.com/scientificreports/ Statistical analyses. The effects of elevated seawater temperature and pCO 2 and their interaction on morphometric components of new shell growth (i.e., clam wet mass gain, shell length gain, shell height gain, shell width gain, and ornamentation width gain) were analyzed using a two-way multivariate analysis of covariance (MANCOVA), with temperature and pCO 2 as fixed factors. As all pre-exposure morphometric variables were strongly, and significantly correlated (Table S2), we used only one of them (i.e., pre-exposure shell length) as a covariate in this initial MANCOVA. As a follow-up to the MANCOVA, a series of two-way ANCOVAs were conducted on each of the five morphometric response variables using the corresponding pre-exposure morphometric trait as the covariate: e.g. pre-exposure shell length for length gain analysis, etc. Unlike morphometric response variables, which were only quantified once per individual at the end of the experiment (i.e., growth over the course of treatment), mineralogical and organic data were collected multiple times for each individual (i.e., sampling of both newly-formed and older-growth shell and scute). For analysis of these data, any measurement with a value less than zero (i.e., a negative elemental concentration indicating a value below the respective limit of detection for that ion) was assigned a value of zero. In addition, measurements with values greater than 10x (mineralogical data) or 5x (organic content data) their respective group mean were removed from analyses as probable technical outliers (N = 18 and N = 1 values removed, respectively). After outlier removal, a total of N = 966 mineralogical and N = 77 organic content values were retained for further analysis.
We then used a series of paired t-tests to determine if there were significant differences in any of the response variables between shell and scute. Because differences were observed between newly-formed shell and newlyformed scute, all mineralogical and organic variables were analyzed separately for shell and scute in subsequent analyses. As explained previously for morphometric data, a similar suite of MANCOVAs (and follow-up ANCO-VAs) were run on the shell organic and mineral content data using post-exposure total wet mass as the covariate.
Before performing MANCOVAs for morphometric, organic, and mineralogical variables, assumptions of normality were visually assessed for all response variables (i.e., five morphometric variables, three organic variables, and nine mineralogical variables) with Q-Q plots. Homogeneity of variance was tested for all variables using Levene's F test. Based on Levene's F test results, the homogeneity of variance assumption was satisfied for all organic variables but was not satisfied for one of the five morphological variables and several of the mineralogical variables (i.e., P < 0.05, see Table S3). Specifically, although the Levene's F tests suggested that the variances associated with the shell width gain morphological variable were not homogenous, an examination of the variances revealed that none of the largest variances were more than four times the size of the corresponding smallest, suggesting that the ANCOVA would be robust to non-homogenous variance in this case 36 . However, for the mineralogical variables, the largest variances were greater than four times the smallest in all cases. Thus, to permit further parametric hypothesis testing, all mineralogical data, except for element/Ca ratios, for both scute and shell were transformed (see below) to satisfy homogeneity of variance assumptions.
For transformation of the mineralogical data, optimization of the Box-Cox parameter lambda (λ) indicated that square-root transformation (λ = 0.5) and log 10 transformation (λ = 0) were roughly equivalent. Thus, we chose to square-root transform all mineralogical variables to avoid complications arising from mineral concentrations equal to zero. Subsequent Levene's F tests analyzing homogeneity of variance on square-root-transformed mineralogical data were non-significant for all minerals (all P > 0.05) save for [ 31 P] and [ 39 K] in newly-formed shell (F 3, 27 = 3.49, P = 0.03, and F 3, 27 = 4.29, P = 0.01, respectively). However, as log-transformation did not improve Levene's F test outcomes for these three variables, square-root-transformed data were used in follow-up parametric models.
For all ANCOVAs, covariates and interactions between the fixed factor and covariates were dropped when not significant. All ANCOVAs were performed using the aov and Anova functions from the "stats" (v. 4.0.2) and "car" (v. 3.0.9) packages, respectively 37 , in the statistical computing software program R (v. 3.6.1) 38 , and results were considered statistically significant (moderate evidence of an effect) and marginally significant (weak evidence of an effect) at alpha values ≤ 0.05 and between 0.05 and 0.1, respectively. Finally, a series of post-hoc analyses (i.e. Bonferroni pairwise comparisons) were performed using estimated marginal means calculated with the R function emmeans in the package "emmeans" (v. 1.5.0) 39 with FDR (i.e. Bonferroni) multiple-comparison p-adjustments to examine individual mean difference comparisons across all levels of experimental treatment. The effect sizes (i.e., partial η 2 ) were calculated using the etaSquared function of the "lsr" package (v. 0.5) 40 and are reported for significant comparisons in Table 2 (shell morphometrics and organic content), Table 3 (mineral content of newly-formed scute/shell), and Table 4 (mineral content of older-growth scute/shell). A summary of all ANCOVA results (including non-significant tests) are provided for morphometrics and organic content (Table S4), mineralogical content in newly-formed scute and shell (Tables S5, S6, respectively) and in oldergrowth scute and shell (Tables S7, S8, respectively).

Results
Shell morphometry. We observed a marginal effect of pCO 2 on aggregate shell morphometry (MAN-COVA, F 5, 23 = 2.53, P = 0.06, Pillai's Trace = 0.35) with increased shell growth rates under elevated pCO 2 . The multivariate effect size (partial η 2 or Pillai's score) of pCO 2 on overall change in shell morphology indicated that 35% of the variance was accounted for by elevated seawater pCO 2 during shell formation. Subsequent ANCOVA analyses for each of the five morphometric response variables revealed a statistically significant effect of pCO 2 on shell growth rates in that variable (Table 2). Effect sizes (partial η 2 ) of pCO 2 ranged from a low of 0.15 (shell height gain) to a high of 0.26 (shell length gain; Table S4).
In older-growth scute, we observed a significant effect of pCO 2 on [ 137 Ba] (ANCOVA, F 1, 23 = 5.00, P = 0.03) and a marginal effect of pCO 2 on [ 88 Sr] (ANCOVA, F 1, 23 = 3.65, P = 0.07). However, subsequent post-hoc tests Table 3. Results of significant (*) and marginally significant (˟) 2-way ANCOVAs and pairwise comparisons (EMMs) examining the effect of elevated seawater temperature and pCO 2 on mineral content of newly-formed scute and shell in the fluted giant clam Tridacna squamosa. Factors with significant (*) or marginal effects (˟) on investigated traits are noted. ANCOVA results for minerals are based on square-root transformed data whereas summary statistics are for non-transformed data. Trait response variable, Factor independent variable including fixed factors and covariate (cov), F F-value, P p-value, η p 2 partial eta squared, N number of samples, M group mean (% change, concentration in mmol kg −1 , or ratio), and SD group standard deviation. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ failed to reveal any differences between pairwise comparisons for either mineral (Bonferroni EMMs post-hoc test, all P > 0.1; Table 4). We observed no other significant or marginal effects of seawater treatments on any other minerals in older-growth scute (ANCOVAs, all P > 0.1; Table S7, Fig. S2a-f).
In older-growth shell, we observed a significant effect of temperature (ANCOVA, F 1, 26 = 6.18, P = 0.02) and a marginal effect of the interaction of temperature and pCO 2 (ANCOVA, F 1, 26 = 5.72, P = 0.02) on [ 24 Mg] with marginally higher [ 24 Mg] under ambient conditions than at elevated temperature (Bonferroni EMMs post-hoc test, t-ratio 1, 26 = 2.49, P = 0.09; Table 4, Fig. S2b). We also observed a significant effect of temperature and of the interaction of pCO 2 and temperature on [ 28 Table 4). We observed a marginal effect of temperature (ANCOVA, F 1, 26 = 3.91, P = 0.06) and a significant interactive effect of temperature and pCO 2 (ANCOVA, F 1, 26 = 4.67, P = 0.04) on [ 40 Ca], but subsequent post-hoc tests failed to reveal any differences between pairwise comparisons for either effect (Bonferroni EMMs post-hoc tests, all P > 0.1; Table 4). There was a marginal effect of the interaction of temperature and pCO 2 (ANCOVA, F 1, 26 = 3.70, P = 0.07) on [ 137 Ba] although subsequent post-hoc tests failed to reveal any differences between pairwise comparisons (Bonferroni EMMs post-hoc test, all P > 0.1; Table 4). Finally, we also observed significant effects of temperature (ANCOVA,  Table 4, Fig. S2e). We observed no other significant or marginal effects of seawater treatments on any other minerals in older-growth shell (ANCOVAs, all P > 0.1; Table S8, Fig. S2a-f). Select Group II elements (i.e., 24 Mg, 88 Sr, and 137 Ba) were also examined as ratios with [ 40 Ca] as the denominator. Because of their similarly-sized ionic radii and electrochemical properties, these cations are known to substitute for 40 Ca within the calcium carbonate matrix of bivalve shells 11,41 with 24 Mg replacing 40 Ca in calcite 42 and 88 Sr and 137 Ba replacing 40 Ca in aragonite 43 . In newly-formed scute, we observed no effect of seawater treatments on any element/Ca ratios (ANCOVAs, all P > 0.1; Table S5). However, in newly-formed shell, we observed a significant effect of elevated temperature (ANCOVA, F 1, 26 = 7.38, P = 0.01) on [ 24 Fig. 4c). In oldergrowth scute, we observed no effect of seawater treatments on any element/Ca ratios (ANCOVAs, all P > 0.1; Table S7, Fig. S3a-c). However, in older-growth shell, we observed a significant effect of elevated temperature (ANCOVA, F 1, 26 = 5. 16 Fig. S3a). We www.nature.com/scientificreports/ observed no effects of seawater treatments on any other element/Ca ratios in older-growth shells (ANCOVAs, all P > 0.1; Table S8, Fig. S3b,c).

Shell organic content.
We observed a marginal effect of elevated temperature (ANCOVA, F 1, 20 = 3.10, P = 0.09) on N-content (weight%) in newly-formed shell. However, subsequent post-hoc testing failed to reveal any differences between pairwise comparisons (Bonferroni EMMs post-hoc test, all P > 0.1; Table 2). We observed no effects of seawater treatments on any other organic content variable in newly-formed shells (ANCOVAs, all P > 0.1; Table S4).

Discussion
Increasing atmospheric pCO 2 is driving reduced carbonate bioavailability and elevated sea surface temperatures (SSTs) throughout the World's oceans. These drivers have the potential to impact shell formation in ecologically and economically important calcifying species such as giant clams. Our results help elucidate the extent to which ocean warming and acidification, alone or in concert, influence shell growth rates and shell mineral and organic content in juvenile Tridacna squamosa giant clams. Overall, we show that alterations in seawater pCO 2 , similar to those projected to occur with the progression of ocean acidification this century, have a stronger influence on shell mineralogy in juvenile T. squamosa than shifts in temperature, projected to occur with ongoing ocean warming, and highlight the importance of seawater pCO 2 (and thus pH) as a driver of not only calcification rates but also of mineral and organic content in giant clam shells.
Shell morphometry and growth. Contrary to our initial hypotheses, we observed no impact of elevated temperature and a positive impact of elevated pCO 2 on shell growth in juvenile T. squamosa. Clams in our study displayed, on average, 2.5x and 1.7x greater gains in shell length and width, respectively, under elevated pCO 2 than under ambient conditions. These results were surprising in light of the majority of previous studies in giant clams that have indicated that elevated SSTs and pCO 2 have positive effects on growth 44 and inhibitory effects on calcification 22,45,46 , respectively. For example, thermally-enhanced growth has been observed in many tridacnid species: including in adult T. squamosa from the Red Sea 44 , as well as in Tridacna squamosina 44 , Tridacna crocea 11,47 , Tridacna derasa 47 , Tridacna maxima 44,47,48 , Tridacna gigas 49 , and Hippopus hippopus 50 . However, detrimental effects have also been observed at extremely elevated SSTs in some instances. In these cases, it is likely that SSTs approached or surpassed ecologically-relevant thermal thresholds resulting in deleterious impacts on growth. For example, in H. hippopus clams, extended exposure to SSTs > 27 °C resulted in a period of erratic growth (i.e., high intraindividual variation) followed by decreased calcification rates 50 . Similarly, a study of gene expression profiles in juvenile T. maxima exposed to SSTs of 32 °C for approx. one week showed that clams upregulated genes involved in the scavenging of reactive oxygen species and in fatty acid rearrangement likely in response to heat stress 18 . Finally, in juvenile T. squamosa, exposure to SSTs > 30 °C for over 40 days led to significantly increased mortality-although low irradiance may also have contributed to this effect 21 . In this study, we did not observe increased mortality under elevated temperature. However juveniles were exposed to relatively severe SST conditions (+ 3 °C over summer maximum temperatures) which may have been at or near their upper thermal limits for growth. This could explain why we observed no thermal-enhancement of shell growth rates.
In contrast to the effects of elevated temperature, previous investigations of the effects of elevated pCO 2 on calcification in giant clams have largely reported decreased shell extension rates under ocean acidification conditions 13,22,45,46,51 . Reduced shell growth rates have been reported previously in juvenile T. squamosa clams (+ 350/1000 µatm for one year 46 and + 585/885 µatm for 10 weeks 51 ) as well as in juveniles of the closely related species T. maxima under elevated pCO 2 (+ 800 μatm for 9 weeks 13 ). However, conflicting evidence exists for T. crocea in which a significant reduction in shell height gain was observed under elevated pCO 2 (+ 600/+ 1600 μatm for 4 weeks), but shell length gain remained unimpacted in the same individuals 23 . Similarly, no effect of pCO 2 on shell growth rate was observed in either T. crocea or T. squamosa clams of the same life stage under similar conditions (+ 350/+ 1000 μatm for one year 46 and + 788 µatm for 6 weeks 52 , respectively). Finally, shell extension rates of juvenile T. derasa clams increased in exposure to high-nutrient, high-pCO 2 conditions 46 . Thus, although the majority of studies report negative effects of elevated pCO 2 on giant clam calcification rates, these effects appear to have been offset in some species under some conditions. In this study, we observed enhanced shell growth rates in juvenile T. squamosa under elevated pCO 2 .
At the molecular level, prolonged exposure of giant clams to elevated pCO 2 reduces net calcification rate presumably as a result of decreased carbonic anhydrase activity 52 . However, elevated pCO 2 can increase calmodulin activity in giant clams 52 , and this calcium-binding protein is increasingly recognized as an important contributor to calcium precipitation in bivalves [52][53][54] . Thus, one possible explanation for the positive effects of ocean acidification on shell growth rates we observed in T. squamosa in this study may be an increase in calmodulin-driven calcium-binding/precipitation under elevated pCO 2 conditions sufficient enough to promote shell growth as has recently been reported in the Pacific oyster Crassostrea gigas 54 .
Another possible explanation for the positive effects of ocean acidification on growth rates in T. squamosa may be a result of indirect fertilization through increased supply of inorganic carbon for symbiont photosynthesis. Peak photosynthetic rates in isolated zooxanthellae maintained under conditions similar to those within the giant clam hemolymph were found to be significantly less than the theoretical maximum, and symbionts, in hospite, are capable of completely depleting inorganic carbon from the host hemolymph 16,55 . This indicates that zooxanthellae are partially carbon limited within their host 56 . In symbiotically-intact giant clams, supply of inorganic carbon to the zooxanthellae is enhanced by the host through an efficient carbon-concentration mechanism 57,58 . Exposure to elevated pCO 2 may therefore increase the rate of carbon supply to the zooxanthellae by elevating levels of www.nature.com/scientificreports/ inorganic carbon in host hemolymph (i.e., tissue acidosis) and increasing export of photosynthetic products to the clam thus fueling host growth. However, this carbon enrichment hypothesis remains equivocal (discussed further in the shell organic content section, below). In addition to the potential for elevated pCO 2 to increase host growth rates, giant clams are also well known to exhibit light-enhanced calcification indicating that increased symbiont photosynthesis also drives increased host biomineralization 11,49,[59][60][61][62] . In this way, giant clams may be able to leverage increased environmental inorganic carbon to promote symbiont photosynthesis, benefiting from increased availability of O 2 and carbohydrates, as well as increased rates of carbonate precipitation. Under this proposed mechanism, exposure to moderately elevated seawater pCO 2 could actually be beneficial for tridacnid clams and could explain the positive effects on growth we report in juvenile T. squamosa clams. However, recent studies in juvenile T. maxima and T. squamosa indicated that exposure to elevated pCO 2 (ca. + 800 µatm) resulted in significant declines in symbiont photosynthetic yield accompanied by reduced zooxanthellae density 13,52 whereas, in juvenile T. crocea, zooxanthellae density increased under elevated pCO 2 but photosynthetic productivity remained constant thus suggesting reduced output per algal cell 23 . Thus, this "fertilization effect" may not be universal across populations, species, and/or environments. For example, the effectiveness of this putative "carbon pump" likely also depends on ambient irradiance levels 22 , as well as on a concomitant increase in the availability of N to the zooxanthellae (see our discussion of shell organic content, below). Differences in rearing conditions, particularly those contributing to photosynthetic productivity, may therefore partially explain the discrepancies in shell growth responses to elevated pCO 2 in giant clams as reported above.

Shell mineralogy. Elemental concentrations. With the exception of [ 137 Ba] and [ 24 Mg] in newly-formed
shell, discussed in the element/Ca section below, and [ 40 Ca] in newly-formed scute, which increased at elevated temperature, mean concentrations of trace elements within T. squamosa scutes or shell were not impacted by exposure to either elevated temperature or pCO 2 (Fig. 3). However, under multistressor conditions, inter-individual variation in trace mineral concentrations within newly-formed shell increased. This increased variability in trace mineral incorporation mirrors the increased intra-individual variation in calcification rates reported in H. hippopus under elevated temperature, which was interpreted as a signal of the onset of thermal stress 50 . Similarly, we hypothesize that the increased variation in trace mineral content we observed in T. squamosa shells formed under multistressor conditions may also indicate the existence of a physiological tolerance threshold after which clams exhibit increased idiosyncrasy in response deriving from different sensitivities between individuals (see our discussion of organismal stress below). This implies that reported variations in mineral and isotopic content of giant clam shells between individuals at the same site 5 may be driven, at least in part, by differential physiological susceptibility to different and/or different combinations of environmental drivers. This observation may have important implications for the use of giant clam carbonates as paleoclimate proxies and for paleoclimate reconstructions, especially when those reconstructions are predicated from one, or a few, individual(s).
Element/Ca ratios. With the exception of [ 75 As], all mineralogical response variables positively covaried under ambient conditions (Fig. S1a). However, the number and strength of these correlations decreased under elevated temperature (Fig. S1b) and elevated pCO 2 (Fig. S1c) in isolation, but not in combination (Fig. S1d). These results suggest a potential antagonistic effect of simultaneous exposure to elevated temperature and pCO 2 on shell trace mineral incorporation, and thus a potential buffering of shell mineral structure under multistressor conditions. Significant attention has been devoted to use of specific element/Ca ratios as paleoclimate-indices-in particular [ 88 (Fig. 4a). In addition, we observed a significant increase in [ 137 Ba]/[ 40 Ca] ratios in newly-formed T. squamosa shell under elevated pCO 2 (Fig. 4b) suggesting an increased substitution of 137 Ba in place of 40 Ca in the CaCO 3 lattice of the shell. To our knowledge, this is the first report of a significant effect of seawater pCO 2 on any element/Ca ratio in giant clams. Because replacement of 40 Ca by other Group II elements has effects on the crystalline ultrastructure of shells 63 , it has the potential to alter their mechanical properties as well. For example, T. squamosa shells with lower CaCO 3 content as a proportion of their weight have been demonstrated to suffer from decreased resistance to crushing forces (i.e., reduced compressive strength) 51 . Thus, the increasing prevalence of 137 Ba we observed in shells at elevated pCO 2 may provide another mechanism for weakening bivalve skeletal structures beyond the effects of reduced calcification and/or shell dissolution generally associated with ocean acidification.
Previous studies have also suggested that increased [ 137 Ba]/[ 40 Ca] in bivalve shells can result from increased dietary input of 137 Ba, for example from increased ingestion of phytoplankton containing 137 Ba 41 . Although we did not measure filtration/clearance rates in this study, experimental aquaria were maintained with pre-filtered seawater and clams were not given any supplemental feedings of plankton. Thus, whereas a shift in the relative balance between photo-and heterotrophy in this species could Table S1).

Shell organic content.
Although it represents only a small fraction of the molluscan shell in terms of weight (typically < 5% in bivalves, ca. 0.9% in Tridacna derasa 75 ), the shell organic matrix plays a significant role in determining shell crystalline microstructure 76 . We observed no effect of exposure to elevated temperature and pCO 2 on C-(total organic and inorganic) or H-content of T. squamosa shells (Fig. 5a,b). However, under multidriver conditions, only a single clam displayed measurable shell organic N after 60 days of exposure (Fig. 5c) and this individual was identified as a data outlier. Analysis of the N-content data without this individual revealed an emerging pattern of decreasing shell organic nitrogen content under elevated temperature conditions in juvenile T. squamosa clams with a potentially synergistic, negative, impact of elevated SST and pCO 2 (Fig. 5c). However, to further test the significance of this trend, a larger number of observations may be needed.
As the organic matrix of tridacnine shell is primarily composed of polysaccharides and glycosylated and unglycosylated proteins and lipids 75 , we postulate that the relative decrease in nitrogen content of shells formed under multistressor conditions may reflect increasing competition for nitrogen between the algal symbiont (as a result of increased primary production from increased inorganic carbon supply) and the host (lipoprotein biosynthesis). There is substantial evidence that symbiotic zooxanthellae are nitrogen-limited in hospite in giant clams and that nitrogen-enrichment and eutrophication can enhance host growth 44,[77][78][79][80][81][82] . Additionally, exposure to elevated seawater pCO 2 (ca. 30 days) was shown to enhance O 2 production in clam-hosted zooxanthellae, and zooxanthellae photosynthetic yield was higher under multistressor conditions (after 30-days exposure) than under ambient conditions thus implying pCO 2 -driven fertilization of giant clam growth 13 . However, conflicting evidence also exists showing reduced photosynthetic yield of clam-hosted zooxanthellae 23 or, alternatively, increased photosynthetic rates per individual zooxanthellae, but overall decreased algal density and net photosynthetic productivity at elevated pCO 2 52 . We postulate that some of this discrepancy regarding the fertilizing effect of elevated pCO 2 on giant clam growth may stem from the interplay of several factors contributing to zooxanthellae photosynthesis in hospite, including irradiance and nutrient supply. For example, if elevated seawater pCO 2 serves as a potential enhanced inorganic carbon source for the algal symbionts in giant clams, as discussed previously, then this would necessitate a concomitant increase in supply of inorganic nitrogen. Given that we see a trend towards nitrogen-depletion in giant clam shells under elevated pCO 2 , especially in combination with elevated temperature, our data may provide additional indirect evidence for increased symbiont productivity under elevated pCO 2 . This in turn ultimately reduces the amount of nitrogen that is available for incorporation   (Fig. 4a). Additionally, we observed increased inter-individual variation in nearly all trace minerals in shells formed under multistressor conditions. Both inter-and intra-individual variation are increasingly recognized as important biological traits underpinning organismal responses to environmental change [83][84][85] and increased variance in elemental composition of giant clam shells has also been proposed as a signature of the onset of thermal stress 50 . However, despite these findings, we saw no evidence for negative impacts of exposure to elevated temperature or pCO 2 on shell formation or animal growth (i.e., mass gain) in juvenile T. squamosa. To the contrary, we observed positive effects of these drivers on shell growth and mass gain in this species. Thus, our data suggest that elevated [ 24 Mg]/[ 40 Ca] in giant clam shells is perhaps best interpreted as the result of a physiological phase shift, potentially signaling the mobilization of compensatory mechanisms (i.e., beneficial plasticity as has been reported in Mytilus mussels 86 ), which may be different or differently modulated in different individuals, rather than the onset of organismal stress sensu stricto.
Interpretation of these data is complex however, especially in light of the fact that element/Ca ratios in giant clam shells can vary significantly between individuals at the same collection site (i.e., high inter-individual variation as described above) 87 and between different regions of the shell either as a result of variation in deposition across life history stages 4,88 or as a result of tissue-specific deposition effects 89 . For example, we observed consistently lower variation in the trace mineral content of scute across treatments suggesting that this region of the exoskeleton is likely formed under different constraints from the shell proper. This dampened response in scutes could reflect selective pressures for maintaining the defensive capabilities in these structures and may reduce their utility as a paleoclimate indices.

Conclusion
We demonstrate that exposure to elevated seawater temperature and pCO 2 altered shell growth rates and composition in juveniles of the fluted giant clam T. squamosa Simultaneous exposure to both drivers resulted in increased inter-individual variation in shell mineral composition and reduced organic N-content, which we hypothesize may signal the onset of physiological stress and/or increased competition for N between the clam host and its algal symbionts. As a consequence, it is increasingly clear that an improved understanding of the physiological mechanisms underpinning shell-formation in these unique, photosymbiotic bivalves is necessary. It remains to be determined, however, whether these effects directly or indirectly impact giant clam survival in the long-term or whether these alterations represent compensatory plasticity meant to maintain overall performance, including potential enhancement of photosymbiont productivity, in the face of multiple stressors. For example, a recent review of the effects of elevated pCO 2 on bivalve shell mineralogy suggests a large capacity for beneficial transgenerational plasticity 90 . Our results suggest the onset of altered physiological states (e.g., shifted heterotrophic-autotrophic balance) in giant clams under projected ocean warming and acidification which may ultimately reduce the protective capacities of their shells and alter exoskeleton element/Ca ratios. This latter finding may have important implications for the use of giant clam carbonates as paleoclimate proxies as we find that changes in seawater temperature and seawater pCO 2 (and/or pH) are capable of driving similar shifts in carbonate element/Ca ratios.